Vitamin B 12

14. Vitamin B 12

14.1 Role of vitamin B 12 in human metabolic processes

Although the nutritional literature still uses the term vitamin B 12 , a more specific name for vitamin B 12 is cobalamin. Vitamin B 12 is the largest of the B complex vitamins, with a relative molecular mass of over 1000. It consists of

a corrin ring made up of four pyrroles with cobalt at the centre of the ring (1, 2). There are several vitamin B 12 -dependent enzymes in bacteria and algae, but no species of plants have the enzymes necessary for vitamin B 12 synthesis. This fact has significant implications for the dietary sources and availability of vitamin B 12 . In mammalian cells, there are only two vitamin B 12 -dependent enzymes (3). One of these enzymes, methionine synthase, uses the chemical form of the vitamin which has a methyl group attached to the cobalt and is called methylcobalamin (see Chapter 15, Figure 15.2). The other enzyme, methylmalonyl coenzyme (CoA) mutase, uses a form of vitamin B 12 that has a 5¢-adeoxyadenosyl moiety attached to the cobalt and is called 5¢-

deoxyadenosylcobalamin, or coenzyme B 12 . In nature, there are two other forms of vitamin B 12 : hydroxycobalamin and aquacobalamin, where hydroxyl and water groups, respectively, are attached to the cobalt. The synthetic form of vitamin B 12 found in supplements and fortified foods is cyanocobalamin, which has cyanide attached to the cobalt. These three forms of vitamin B 12 are enzymatically activated to the methyl- or deoxyadenosylcobalamins in all mammalian cells.

14.2 Dietary sources and availability

Most microorganisms, including bacteria and algae, synthesize vitamin B 12 , and they constitute the only source of the vitamin (4). The vitamin B 12 syn- thesized in microorganisms enters the human food chain through incorpora- tion into food of animal origin. In many animals, gastrointestinal fermentation

supports the growth of these vitamin B 12 synthesizing microorganisms, and subsequently the vitamin is absorbed and incorporated into the animal tissues. This is particularly true for the liver, where vitamin B 12 is stored in large con-

VITAMIN AND MINERAL REQUIREMENTS IN HUMAN NUTRITION

centrations. Products from herbivorous animals, such as milk, meat, and eggs, thus constitute important dietary sources of the vitamin, unless the animal is subsisting in one of the many regions known to be geochemically deficient in cobalt (5). Milk from cows and humans contains binders with very high affin-

ity for vitamin B 12 , though whether they hinder or promote intestinal absorp- tion is not entirely clear. Omnivores and carnivores, including humans, derive dietary vitamin B 12 almost exclusively from animal tissues or products (i.e. milk, butter, cheese, eggs, meat, poultry). It appears that the vitamin B 12 required by humans is not derived from microflora in any appreciable quan- tities, although vegetable fermentation preparations have been reported as

being possible sources of vitamin B 12 (6).

14.3 Absorption

The absorption of vitamin B 12 in humans is complex (1, 2). Vitamin B 12 in food is bound to proteins and is only released by the action of a high concentration of hydrochloric acid present in the stomach. This process results in the free form of the vitamin, which is immediately bound to a mixture of glycoproteins secreted by the stomach and salivary glands. These

glycoproteins, called R-binders (or haptocorrins), protect vitamin B 12 from chemical denaturation in the stomach. The stomach’s parietal cells, which secrete hydrochloric acid, also secrete a glycoprotein called intrinsic factor.

Intrinsic factor binds vitamin B 12 and ultimately enables its active absorption. Although the formation of the vitamin B 12 –intrinsic factor complex was ini- tially thought to happen in the stomach, it is now clear that this is not the case. At an acidic pH, the affinity of the intrinsic factor for vitamin B 12 is low whereas its affinity for the R-binders is high. When the contents of the stomach enter the duodenum, the R-binders become partly digested by the

pancreatic proteases, which in turn causes them to release their vitamin B 12 . Because the pH in the duodenum is more neutral than that in the stomach, the intrinsic factor has a high binding affinity to vitamin B 12 , and it quickly binds the vitamin as it is released from the R-binders. The vitamin

B 12 –intrinsic factor complex then proceeds to the lower end of the small intestine, where it is absorbed by phagocytosis by specific ileal receptors (1, 2).

14.4 Populations at risk for, and consequences of, vitamin

B 12 deficiency

14.4.1 Vegetarians

Because plants do not synthesize vitamin B 12 , individuals who consume diets completely free of animal products (vegan diets) are at risk of vitamin B 12 defi-

14. VITAMIN B 12

ciency . This is not true of lacto-ovo vegetarians, who consume the vitamin in eggs, milk, and other dairy products.

14.4.2 Pernicious anaemia

Malabsorption of vitamin B 12 can occur at several points during digestion (1, 4). By far the most important condition resulting in vitamin B 12 malabsorp- tion is the autoimmune disease called pernicious anaemia (PA). In most cases of PA, antibodies are produced against the parietal cells causing them to atrophy, and lose their ability to produce intrinsic factor and secrete hydrochloric acid. In some forms of PA, the parietal cells remain intact but autoantibodies are produced against the intrinsic factor itself and attach to it,

thus preventing it from binding vitamin B 12 . In another less common form of PA, the antibodies allow vitamin B 12 to bind to the intrinsic factor but prevent the absorption of the intrinsic factor–vitamin B 12 complex by the ileal recep- tors. As is the case with most autoimmune diseases, the incidence of PA increases markedly with age. In most ethnic groups, it is virtually unknown to occur before the age of 50, with a progressive rise in incidence thereafter (4). However, African American populations are known to have an earlier age of presentation (4). In addition to causing malabsorption of dietary vitamin

B 12 , PA also results in an inability to reabsorb the vitamin B 12 which is secreted in the bile. Biliary secretion of vitamin B 12 is estimated to be between 0.3 and 0.5mg/day. Interruption of this so-called enterohepatic circulation of vitamin

B 12 causes the body to go into a significant negative balance for the vitamin. Although the body typically has sufficient vitamin B 12 stores to last 3–5 years, once PA has been established, the lack of absorption of new vitamin B 12 is compounded by the loss of the vitamin because of negative balance. When the stores have been depleted, the final stages of deficiency are often quite rapid, resulting in death in a period of months if left untreated.

14.4.3 Atrophic gastritis

Historically, PA was considered to be the major cause of vitamin B 12 defi- ciency, but it was a fairly rare condition, perhaps affecting between one and

a few per cent of elderly populations. More recently, it has been suggested that a far more common problem is that of hypochlorhydria associated with atrophic gastritis, where there is a progressive reduction with age of the ability of the parietal cells to secrete hydrochloric acid (7). It is claimed that perhaps up to one quarter of elderly subjects could have various degrees of hypochlorhydria as a result of atrophic gastritis. It has also been suggested that bacterial overgrowth in the stomach and intestine in individuals suffer-

ing from atrophic gastritis may also reduce vitamin B 12 absorption. The

VITAMIN AND MINERAL REQUIREMENTS IN HUMAN NUTRITION

absence of acid in the stomach is postulated to prevent the release of protein- bound vitamin B 12 contained in food but not to interfere with the absorption of the free vitamin B 12 found in fortified foods or supplements. Atrophic gas- tritis does not prevent the reabsorption of biliary vitamin B 12 and therefore does not result in the negative balance seen in individuals with PA. Nonethe- less, it is agreed that with time, a reduction in the amount of vitamin B 12

absorbed from the diet will eventually deplete vitamin B 12 stores, resulting in overt deficiency. When considering recommended nutrient intakes (RNIs) for vitamin B 12 for the elderly, it is important to take into account the absorption of vitamin

B 12 from sources such as fortified foods or supplements as compared with dietary vitamin B 12 . In the latter instances, it is clear that absorption of intakes of less than 1.5–2.0mg/day is complete—that is, for daily intakes of less than 1.5–2.0mg of free vitamin B 12 , the intrinsic factor-mediated system absorbs that entire amount. It is probable that this is also true of vitamin B 12 in forti- fied foods, although this has not been specifically examined. However, absorption of food-bound vitamin B 12 has been reported to vary from 9% to 60% depending on the study and the source of the vitamin, which is perhaps related to its incomplete release from food (8). This has led many to estimate absorption as being up to 50% to correct for the bioavailability of vitamin B 12 from food.

14.5 Vitamin B 12 interaction with folate or folic acid

One of the vitamin B 12 -dependent enzymes, methionine synthase, functions in one of the two folate cycles, namely, the methylation cycle (see Chapter 15). This cycle is necessary to maintain availability of the methyl donor, S-adenosylmethionine. Interruption of the cycle reduces the level of S-adeno-

sylmethionine. This occurs in PA and other causes of vitamin B 12 deficiency, producing as a result demyelination of the peripheral nerves and the spinal column, giving rise to the clinical condition called subacute combined degen- eration (1, 2). This neuropathy is one of the main presenting conditions in PA. The other principal presenting condition in PA is a megaloblastic anaemia morphologically identical to that seen in folate deficiency. Disruption of the methylation cycle also causes a lack of DNA biosynthesis and anaemia.

The methyl trap hypothesis is based on the fact that once the cofactor 5,10- methylenetetrahydrofolate is reduced by its reductase to form 5-methylte- trahydrofolate, the reverse reaction cannot occur. This suggests that the only way for the 5-methyltetrahydrofolate to be recycled to tetrahydrofolate, and thus to participate in DNA biosynthesis and cell division, is through the

vitamin B 12 -dependent enzyme methionine synthase. When the activity of this

14. VITAMIN B 12

synthase is compromised, as it would be in PA, the cellular folate will become progressively trapped as 5-methyltetrahydrofolate (see Chapter 15, Figure 15.2). This will result in a cellular pseudo-folate deficiency where, despite adequate amounts of folate, anaemia will develop, which is identical to that seen in true folate deficiency. Clinical symptoms of PA, therefore, include

neuropathy, anaemia, or both. Treatment with vitamin B 12 , if given intramus- cularly, will reactivate methionine synthase, allowing myelination to restart. The trapped folate will be released and DNA synthesis and generation of red cells will cure the anaemia. Treatment with high concentrations of folic acid will treat the anaemia but not the neuropathy of PA. It should be stressed that the so-called “masking” of the anaemia of PA is generally agreed not to occur at concentrations of folate found in food or at intakes of the synthetic form of folic acid at usual RNI levels of 200 or 400mg/day (1). However, there is some evidence that amounts less than 400mg may cause a haematologic response and thus potentially treat the anaemia (9). The masking of the anaemia definitely occurs at high concentrations of folic acid (>1000mg/day). This becomes a concern when considering fortification with synthetic folic acid of a dietary staple such as flour (see Chapter 15).

In humans, the vitamin B 12 -dependent enzyme methylmalonyl CoA mutase functions both in the metabolism of propionate and certain amino acids—converting them into succinyl CoA—and in the subsequent metabo- lism of these amino acids via the citric acid cycle. It is clear that in vitamin

B 12 deficiency the activity of the mutase is compromised, resulting in high plasma or urine concentrations of methylmalonic acid (MMA), a degradation product of methylmalonyl CoA mutase. In adults, this mutase does not appear to have any vital function, but it clearly has an important role during embryonic life and in early development. Children deficient in this enzyme, through rare genetic mutations, suffer from mental retardation and other developmental defects.

14.6 Criteria for assessing vitamin B 12 status

Traditionally it was thought that low vitamin B 12 status was accompanied by

a low serum or plasma vitamin B 12 level (4). Recently, Lindenbaum et al. (10) challenged this assumption, by suggesting that a proportion of people with normal serum and plasma vitamin B 12 levels are in fact vitamin B 12 deficient. They also suggested that elevation of plasma homocysteine and plasma MMA are more sensitive indicators of vitamin B 12 status. Although plasma homo- cysteine can also be elevated because of folate or vitamin B 6 deficiency, elevation of MMA apparently always occurs with poor vitamin B 12 status. However, there may be other reasons why MMA is elevated, such as renal

VITAMIN AND MINERAL REQUIREMENTS IN HUMAN NUTRITION

insufficiency, so the elevation of MMA, in itself, is not diagnostic. Thus, low serum or plasma levels of vitamin B 12 should be the first indication of poor status and this could be confirmed by an elevated MMA if this assay was available.

14.7 Recommendations for vitamin B 12 intakes

The Food and Nutrition Board of the National Academy of Sciences (NAS) Institute of Medicine (8) has recently conducted an exhaustive review of the

evidence regarding vitamin B 12 intake, status, and health implications for all age groups, including the periods of pregnancy and lactation. This review has lead to calculations of what they have called an estimated average requirement (EAR), which is defined by NAS as “the daily intake value that is estimated to meet the requirement, as defined by the specific indicator of adequacy, in half of the individuals in a life-stage or gender group” (8). The NAS then

estimated a recommended dietary allowance (RDA) for vitamin B 12 , as this daily intake value plus 2 standard deviations (SDs).

Some members of the present FAO/WHO Consultation were involved in the preparation and review of the NAS recommendations and judge them to

be the best estimates currently available. The FAO/WHO Consultation thus felt it appropriate to adopt the same approach used by the NAS in deriving the RNIs for vitamin B 12 . Therefore, the EARs given in Table 14.1 are the same as those proposed by the NAS, and the RNIs (which are equivalent to

TABLE 14.1

Estimated average requirements (EARs) and recommended nutrient intakes (RNIs) for vitamin

B 12 , by group

Group

EAR (mg/day)

RNI (mg/day)

Infants and children 0–6 months

Adolescents 10–18 years

Adults 19–65 years

Pregnant women

Lactating women

Source: adapted from reference (8).

14. VITAMIN B 12

the RDAs used by the NAS) calculated as the EAR plus 2 SD. Supporting evidence for the recommendations for each age group is summarized below.

14.7.1 Infants

As with other nutrients, the principal way to determine requirements of infants is to examine the levels in milk from mothers on adequate diets. There

is a wide difference in the vitamin B 12 values reported in human milk because of differences in methodology. The previous FAO/WHO Expert Consulta- tion (11) based their recommendations on milk vitamin B 12 values of normal women of about 0.4mg/l. For an average milk production of 0.75l/day, the vitamin B 12 intake by infants would be 0.3mg/day (12). Other studies have reported concentrations of vitamin B 12 in human milk in the range 0.4–0.8mg/l (13–17). Although daily intakes ranging from 0.02 to 0.05mg/day have been found to prevent deficiency (18, 19), these intakes are totally inadequate for long-term health. Thus, based on the assumption that human milk contains

enough vitamin B 12 for optimum health, an EAR between 0.3 and 0.6mg/day seems reasonable giving an RNI of between 0.4 and 0.7mg/day. It would seem appropriate to use the lower RNI figure of 0.4mg/day for infants aged 0–6 months and the higher RNI figure of 0.7mg/day for infants aged 7–12 months (Table 14.1).

14.7.2 Children

The Food and Nutrition Board of the NAS Institute of Medicine (8) sug- gested the same intakes for adolescents as those for adults (see section 14.7.3) with progressive reduction of intake for younger groups.

14.7.3 Adults

Several lines of evidence point to an adult average requirement of about 2.0mg/day. The amount of intramuscular vitamin B 12 needed to maintain remission in people with PA suggests a requirement of about 1.5mg/day (10), but they would also be losing 0.3–0.5mg/day through interruption of their enterohepatic circulation. This might suggest a requirement of 0.7–1.0mg/day

for those without PA. Because vitamin B 12 is not completely absorbed from food, an adjustment of 50% has to be added, giving a range of 1.4–2.0mg/day (4). Therapeutic response to dietary vitamin B 12 suggests a minimum requirement of something less than 1.0mg/day (8), which again suggests a requirement of 2.0mg/day, allowing for the conservative correction that

only half of dietary vitamin B 12 is absorbed (8). Diets containing 1.8mg/day seemed to maintain adequate status but intakes lower than this resulted in subjects showing some signs of deficiency (8). Furthermore, dietary intakes

VITAMIN AND MINERAL REQUIREMENTS IN HUMAN NUTRITION

of less than 1.5mg/day were reported to be inadequate in some subjects (20).

In summary, the average requirement could be said to be 2mg/day (8).

Assuming the variability of the requirements for vitamin B 12 is accounted for by adding 2 SDs, the RNI for adults and the elderly becomes 2.4mg/day.

14.7.4 Pregnant women

The previous FAO/WHO Expert Consultation (11) estimated that 0.1–0.2mg/day of vitamin B 12 is transferred to the fetus during the last two trimesters of pregnancy. On the basis of fetal liver content from postmortem samples (21–23), there is further evidence that the fetus

accumulates, on average, 0.1–0.2mg/day of vitamin B 12 during pregnancies of women with diets which provide adequate levels of vitamin B 12 . It has been reported that children born to vegetarians or other women with a low vitamin B 12 intake subsequently develop signs of clinical vitamin B 12 deficiency such as neuropathy (13). Therefore, in order to derive an EAR for pregnant women, 0.2mg/day of vitamin B 12 was added to the EAR for adults, to give an EAR of 2.2mg/day and a RNI of 2.6mg/day during pregnancy.

14.7.5 Lactating women

It is estimated that 0.4mg/day of vitamin B 12 is found in the human milk of women with adequate vitamin B 12 status (8). Therefore, an extra 0.4mg/day of vitamin B 12 is needed during lactation in addition to the normal adult require- ment of 2.0mg/day, giving a total EAR of 2.4mg/day and a RNI of 2.8mg/day during lactation.

14.8 Upper limits

The absorption of vitamin B 12 mediated by the glycoprotein, intrinsic factor, is limited to 1.5–2.0mg per meal because of the limited capacity of the receptors. In addition, between 1% and 3% of any particular oral adminis-

tration of vitamin B 12 is absorbed by passive diffusion. Thus, if 1000mg vitamin B 12 (sometimes used to treat those with PA) is taken orally, the amount absorbed would be 2.0mg by active absorption plus up to about 30 m g by passive diffusion. Intake of 1000mg vitamin B 12 has never been reported to have any side-effects (8). Similar large amounts have been used in some preparations of nutritional supplements without apparent ill effects. However, there are no established benefits for such amounts. Such high intakes thus rep- resent no benefit in those without malabsorption and should probably be avoided.

14. VITAMIN B 12

14.9 Recommendations for future research

Because they do not consume any animal products, vegans are at risk of vitamin B 12 deficiency. It is generally agreed that in some communities the only source of vitamin B 12 is from contamination of food by microorganisms. When vegans move to countries where standards of hygiene are more strin- gent, there is good evidence that risk of vitamin B 12 deficiency increases in adults and, particularly, in children born to and breastfed by women who are strict vegans.

As standards of hygiene improve in developing countries, there is a concern

that the prevalence of vitamin B 12 deficiency might increase. This should be ascertained by estimating plasma vitamin B 12 levels, preferably in conjunction with plasma MMA levels in representative adult populations and in infants. Further research needs include the following:

• ascertaining the contribution that fermented vegetable foods make to the vitamin B 12 status of vegan communities; • investigating the prevalence of atrophic gastritis in developing countries to determine its extent in exacerbating vitamin B 12 deficiency.

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